Overall, the expertise of the Mass Spectrometry Unit is being widely used to further the research of multiple groups within the NIH. In FY2019, the unit collaborated in 67 different projects, with more than 3000 samples processed and analyzed. These projects are being performed in collaboration with 44 different investigators. Among these are projects to characterize the post-translational modifications of target proteins, including sites of phosphorylation, ubiquitination, acetylation, and methylation, to better understand signal transduction, protein regulation, and the effects of small molecule inhibitors. The resource is also being used to identify protein interactors of both proteins and nucleic acids, including identification of those that change following post-translational modification. Mass spectrometry is additionally being used extensively for large-scale quantitative proteomics projects, using both isotopic labeling and label-free approaches. Structural mass spectrometry applications, such as crosslinking and limited proteolysis methods, are being used to investigate protein conformation. Finally, the resource is using inductively-coupled plasma mass spectrometry (ICP-MS) to quantify the level of metals in biological samples, including copper, iron, lead, tungsten, and platinum. In the past year, six collaborative studies have been published; several other projects are nearing completion or manuscripts are under review. With Dr. Carole Parent, formerly of the Laboratory of Cellular and Molecular Biology, we used mass spectrometry to investigate the composition of extracellular vesicles released by Dictyostelium discoideum for signaling purposes. Using label-free quantitative mass spectrometry, we analyzed the composition of the extracellular vesicles released by ACA WT and ACA-YFP/aca- cells. Comparing the samples, we found that approximately 60% of the identified proteins were present in both samples, indicating that the overexpression of ACA-YFP did not dramatically change the protein makeup of the secreted vesicles. GO network analysis revealed significant enrichment in biological processes related to the cytoskeleton, vesicular trafficking, signaling, energy metabolism, and translation. In addition, 13 ABC transporters were identified in the extracellular vesicles, and further experiments demonstrated that the ABCC8 transporter was responsible for release of cAMP produced by ACA in the vesicles. This research was published in the Journal of Cell Biology. The tuberous sclerosis complex (TSC) is a negative regulator of the mTOR complex 1, a signaling node promoting cellular growth in response to various nutrients and growth factors. Using pulldown and mass spectrometry approaches, we identified the TSC complex member TBC1 domain family member 7 (TBC1D7) as a novel binding partner for PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), a negative regulator of Akt kinase signaling. Sequence analysis identified a putative site for both Akt-mediated phosphorylation and 14-3-3 binding at Ser124, and we used mass spectrometry to demonstrate that Akt phosphorylates TBC1D7 at Ser124. Further experiments showed that this phosphorylation stabilized TBC1D7. Overall, our studies revealed that Akt activity determines the phosphorylation status of TBC1D7 at the phospho-switch Ser124, which governs binding to either 14-3-3 or beta-TrCP2, resulting in increased or decreased stability of TBC1D7, respectively. This research, performed in collaboration with Dr. Michael Gottesman, Laboratory of Cell Biology, was published in the Journal of Biological Chemistry. In the third published project, binding partners of ARAP2, an Arf GTPase-activating protein (Arf GAP) composed of a SAM, 5 PH, Arf GAP, Ank repeat, Rho GAP and Ras Association domains that specifically uses Arf6 as a substrate, were investigated by mass spectrometry. ARAP2 is known to localize to and regulate focal adhesions (FA). In this research, performed in collaboration with Dr. Paul Randazzo, Laboratory of Cellular and Molecular Biology, we examined the mechanism by which ARAP2 affects focal adhesions. We found that ARAP2 control of FA dynamics was dependent on its enzymatic Arf GAP activity. In addition, in some cells, ARAP2 regulated phosphoAkt (pAkt) levels, but ARAP2 control of FAs did not require Akt and conversely, the effects on pAkt were independent of FAs. Furthermore, the effect of ARAP2 on Akt did not require Arf GAP activity, which is necessary for effects on FAs and integrin traffic. Mass spectrometry analysis revealed non-muscle myosin IIA, tubulin, moesin and alpha-actinin as potential binding partners of ARAP2. The results of this study were published in Biology of the Cell. In collaboration with Dr. Yves Pommier, Developmental Therapeutics Branch, mass spectrometry was used to identify binding partners of mitochondrial topoisomerase IB, a nuclear-encoded topoisomerase that is exclusively localized to mitochondria and which resolves topological stress generated during mtDNA replication and transcription. Nearly half of the detected proteins were involved in mitochondrial translation and constituents of the mitoribosome. The association of TOP1MT with mitochondrial translation was also reflected in gene ontology enrichment analysis, showing significant scores for processes associated with mitochondrial translation, rRNA processing, and cell redox homeostasis. These interactions ensure optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The results of this study were published in Nature Communications. The mass spectrometry resource was also able to contribute mass spectrometry to the NIH Extracellular RNA Communication Consortium effort to develop a map of cell-cell communication mediated by extracellular RNA. Mass spectrometry was used to characterize the protein composition of extracellular vesicles isolated from human serum and plasma fractions. Pathway enrichment analysis of the mass spectrometry results revealed protein enrichments consistent with extracellular vesicles: in the biological process category, exocytosis and secretion pathways were detected, whereas in the cellular component category, membrane-bounded vesicles, extracellular exosomes, and organelle lumen-parts were detected. These results helped to characterize the cargo types detected in human samples. This study, performed in collaboration with Dr. Jennifer Jones, Laboratory of Pathology, was published in Cell. Working with Dr. Daniel Appella, NIDDK, mass spectrometry was used to determine the mechanism of action of a novel class of inhibitors of the HIV Gag polyprotein. Previously, we that the inhibitors covalently modify the zinc-binding domains of the NC domain of Gag in vitro and in cells. In further work, we used mass spectrometry to identify multiple sites of covalent modification throughout the Gag polyprotein due to inhibitor reaction. The earliest reactions occurred within matrix and NC domains, with additional sites observed at later times. Similar patterns of modification were observed using in vitro reactions and in Gag isolated from virions released by treated cells. These covalent reactions inhibit maturation and prevent formation of infectious viral particles. The results of this work were published in the Journal of the American Chemical Society.